Isolation and identification of a stabilized derivative of aldophosphamide, a major metabolite of cyclophosphamide.

homoalcophosphamide by the procedure of Corey and Kim (7) gave diethylaldophosphamide and diethylhomoaldophos phamide, respectively (Chart 2). Diethylaldophosphamide was characterized by mass spectral analysis [m/e 283, 1 Cl, M2— CH2C1@; m/e 192, no Cl, M —N(CH2CH2C1)2] and by positive qualitative tests as an aldehyde and an alkylating agent. Complete purification of diethylaldophosphamide by TLC [RF 0.56 in chloroform:butanol (9: 1)] at 5°failed because of its instability. Diethylhomoaldophosphamide was characterized as follows: IR (cm' ): 2970, 2935, 2895, 1715 (C=O), 1450, 1380,1345, 1220,1195,1130,1085,1030, 975, 950, 915, 790, 750, 715, 650, 540; PMRCDC13, tetramethylsilane reference:@ 1.0 to 1.3 (6H, triplet, Cl3—), 1.84 to 2.20 (2H, multiplet, —OCH2CH@—), 2.50 to 2.70 [21, doublet of triplets, CH@—C(O)—] , 2.88 to 3.80(12H multiplet, CH@@Cl and @@CH3), 3.88 to 4.12 (211, quartet, —0—c5!:!z —)†̃ 9.78 to 9.86 (1H, multiplet, HCO): mass spectrum (m/e): 331 (2 Cl, M —Cl3), 303 (2 Cl, M — CH2CHO), 297 (1 Cl, M —CI2C1), 206 [no Cl, M — N(CH2CI2C1)2 base peak] ; TLC : RF 0.65 in acetone:chloro form (1:1), RF 0.40 in acetone:chloroform (1 :4). Diethylalco phosphamide and diethyihomoalcophosphamide were synthe sized from bis(2-chloroethyl)phosphoraimdic dichlonde (8) by successive treatment with I equivalent each of diethylamine and of triethylamine followed by 1 equivalent each of 1,3-propanediol or 1,4-butanediol and of triethylamine. Purification was accomplished by TLC in acetone:chloroform (1 : 1) and chloroform :methanol (9: 1), respectively. TLC. TLC analysis was performed on Analtech (Newark, Del.) precoated Silica Gel G plates (250 jim thick). The plates were activated at 120°for 1 hr and stored in a dessicated chamber. Alkylating Activity. Thin-layer chromatograms were sprayed with a 1% solution of 4-(p-nitrobenzyl)pyridine in acetone, heated in an oven for 15 mm at 140°,and sprayed with a 3% solution of potassium hydroxide in methanol. Alkylating components yielded blue spots. Instrumental Analysis. Mass spectral analysis was performed with a Hitachi Model RMU-6D mass spectrometer, IR analysis with a Perkin-Elmer Model 621 spectrophotometer, and PMR analysis with a Varian XL-l00-lS spectrometer.